Variation in the tonoplast cadmium transporter heavy metal ATPase 3 (HMA3) homolog gene in Aegilops tauschii

The functionality of HMA3 is a key determinant controlling Cd accumulation in the shoots and grains of plants. Wild relatives of modern crop plants can serve as sources of valuable genetic variation for various traits. Here, resequencing of HMA3 homoeologous genes from Aegilops tauschii (the donor of the wheat D genome) was carried out to identify natural variation at both the nucleotide and polypeptide levels. HMA3 homoeologs are highly conserved, and 10 haplotypes were revealed based on 19 single nucleotide polymorphisms (eight induced single amino acid residue substitutions, including 2 altered amino acids in transmembrane domains) in 80 widely distributed Ae. tauschii accessions. The results provide genetic resources for low/no Cd concentration wheat improvement.


Introduction
The low cadmium (Cd) concentration trait in bread wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42) grains is important in terms of food safety because bread wheat is a crucial source of calories, accounting for 20% of calories for human consumption and feeding more than 35% of the world's population [1]. Cadmium can accumulate in the human body over time as a result of the ingestion of food containing Cd, leading to a risk of chronic toxicity with excessive intake [2]. Furthermore, Cd is not phytotoxic at the low concentrations that are of concern for human health [3]. Therefore, reducing Cd accumulation in wheat grains is essential for food safety and human health issue.
The tonoplast transporter HMA3 (P 1B -type of heavy metal ATPases 3) plays an important role in the transport and homeostasis of metals in plants [4]. Loss of function of HMA3 leads to decreased Cd sequestration in the roots but greatly increased translocation of Cd from roots to shoots in Arabidopsis thaliana and rice [5][6][7][8]. In contrast, overexpression of OsHMA3 increases Cd sequestration in the roots and Cd tolerance, and markedly decreases Cd translocation to the shoots and Cd concentration in the grains (decreased Cd concentration by 94-98%) [9,10]. Overexpression of rice OsHMA3 in wheat also greatly decreases Cd translocation from the roots to the shoots and Cd accumulation in wheat grains [11]. Recently, TdHMA3-B1 (Cdu-B1) was identified as a QTL located on the long arm of chromosome 5B that explains >80% of the phenotypic variation in grain Cd concentration in tetraploid durum wheat (T. durum, AABB, 2n = 4x = 28) [12][13][14][15][16]. Cd associated SNP markers on 5AL were identified in a region homoeologous to the TdHMA3-B1 locus on 5BL in durum wheat, which explained 12%-19% of variation in grain Cd concentration depending on the environment in bread wheat [17]. A 17-bp duplication in the first exon that creates two alternative alleles, TdHMA3-B1a (the functional allele being able to transport Cd into the vacuoles with low grain Cd) and TdHMA3-B1b (the nonfunctional allele being inactive with high grain Cd), in durum wheat [16]. In addition, the high-cadmium allele is widespread in durum wheat cultivars but undetected in wild emmer accessions, the frequency of the high-cadmium allele are increased from domesticated emmer accessions to modern durum wheat [16]. These studies indicate that the functionality of HMA3 is a key determinant controlling Cd accumulation in the shoots and grains of plants and that wild relatives of modern crop plants can serve as sources of valuable genetic diversity to develop low/no Cd concentration cultivars.
Aegilops tauschii (DD, 2n = 2x = 14), a wild relative of wheat, is the D genome progenitor of hexaploid wheat [18][19][20][21]. It has been shown to be an effective medium for transferring valuable genetic variation from Ae. tauschii to common wheat [22][23][24]. The addition of the D genome changed wheat from tetraploid to hexaploid. Indeed, hexaploid wheat became a major food crop worldwide only after the addition of the D genome [25]. It can be seen that their ability to spread widely may be related to the great adaptability brought by the D genome, which also explains why they have many agronomic valuable characteristics, including tolerance to drought [26], salt [27], low phosphorus [28] and extreme environments. Besides that, The Cdtolerant Ae. tauschii accessions were indentified by Genome-wide association analysis, which may be used as germplasm resources of wheat breeding for no or low Cd content [29]. Here, we report a survey of the natural variation in potential Ae. tauschii germplasm for HMA3 homoeologous sequences across a diverse geographical distribution and provide genetic resources for low/no Cd concentration wheat improvement.

Plant materials
The Ae. tauschii germplasm panel consisted of 80 accessions ( Table 1). The materials were planted at the Wenjiang Experimental Station of Sichuan Agricultural University in Chengdu, China. All materials were stored at the Triticeae Research Institute, Sichuan Agricultural University.
Accessions prefixed with either PI and CIae were provided by the USDA-ARS, those with KU by the Japanese National BioResource Project (NBRP), those with IG by the International Center for Agricultural Research in Ardid Areas (ICARDA), those with TA by Kansas State University, those with either K, AL and RL by UC Davis, and those with AS by Sichuan Agricultural University Triticeae Research Institute. Genotype (Sublineage) were previously published [30].

PLOS ONE
Variation in HMA3 homoeologs in Aegilops tauschii 10 min. The cycling conditions for fragment 2 were initial denaturation at 95˚C for 5 min followed by 35 cycles of 94˚C for 40 s, annealing at 61˚C for 40 s, and extension at 72˚C for 2 min, with a final extension at 72˚C for 10 min. The amplified products were separated on a 1.0% agarose gel in 1×TAE buffer (0.04 mol/L Tris base, 0.02 mol/L acetic acid, and 1.0 mmol/ L EDTA) followed by staining with ethidium bromide. The PCR products were sequenced directly by Qingke (Chengdu, China).

Sequence alignment and phylogenetic analysis
Multiple sequence alignments at both the nucleotide and predicted polypeptide levels were performed using DNAMAN v6.0 software (Lynnon Biosoft, Quebec, Canada). The transmembrane domains were predicted using TOPCONS (https://topcons.net/pred/result/rst_ yGKZeW/). Phylogenetic trees were constructed based on the neighbor-joining method using MEGA v11 software [31]. Bootstrap analysis was based on 1,000 replicates.

Ae. tauschii HMA3-D1: Peptide sequence polymorphism
At the polypeptide sequence level, the 10 haplotypes from Ae. tauschii collapsed into ten distinct polypeptides. Polypeptide sequence alignment of HMA3 homoeologs is shown in Fig 2. The predicted protein of TaHMA3-D1 (AIA57682.1) in CS was indistinguishable from Hap-8. Their structural organization shared conserved transmembrane domains (TM1 to 8, boxed in Fig 2). Two of the eight altered amino acids were found in transmembrane domains. One was different from that of bread wheat, tetraploid wheat, barley, rice, maize, and A. thaliana at position 134, from A to V, in TM2, which was present in Hap-4 and -7 of Ae. tauschii, while the other was an A to T change at position 178 in TM4 in Hap-2, -4 and -7 (Fig 2). The remaining 6 altered peptide sequences were at position 306, from R to S, in Hap-1 and -6; position 475, from Q to E, and position 524, from Y to C, in Hap-1 to -4, -6 and -10; position 656, from A to V, in Hap-1 to -4 and -6; position 726, from A to V, in Hap-1 to -4 and -10; and position 811, from P to S, in Hap-1 to -10, except for Hap-9 (Fig 2).

HMA3-D1 homoeolog phylogeny
The phylogeny of the protein sequences of wheat HMA3 homoeologs is shown in Fig 3. Each of the three homoeologs from Ae. tauschii and Triticum formed a distinct clade. The homoeologs from the B and D genome clades appeared to be more closely related to one another than to the homoeologs from the A genome clade. Within the homoeologs from the D genome clade, two subgroups were recognizable: one comprised the Ae. tauschii sequences Hap-1 to -4, -6 and -10, and the other comprised the D genome sequences present in Ae. tauschii Hap-5, -7 to -9 and T. aestivum. The cluster of cereal sequences, including Ae. tauschii, Triticum, Hordeum, Brachypodium, Oryza, Zea and Sorghum, showed that they were phylogenetically closely related to one another. The HMA3 homologous sequences of Arabidopsis thaliana (AT4G30120) as the outgroup.

Discussion
Previous studies showed that OsHMA3 from a low Cd-accumulating cultivar limits the translocation of Cd from the roots to the above ground tissues by selectively sequestrating Cd into the root vacuoles [5,32]. Allelic variations in the coding sequences of this gene were responsible for the loss of function, such as a mutation at the 80th amino acid (R80H) [5], a mutation at the 380th amino acid in TM6 from Ser to Arg [7], a 153-bp (51-aa) deletion [6], and 14-aa deletion [8]. A strong association between the BrHMA3 haplotypes and the Cd translocation phenotypes was also found, and variation in the BrHMA3 coding sequence is a key determinant of Cd translocation to and accumulation in the leaves of B. rapa [33]. In durum wheat, the TdHMA3-B1 (Cdu-B1) gene explained approximately 80% of the variation in grain Cd concentration [14][15][16]. The 17-bp duplication was responsible for the loss of function [16]. The Cd-associated SNPs identified in bread wheat are in a region of 5AL that is homoeologous to the region of Cdu-B1 in durum [17].
The diverse Ae. tauschii offers a valuable gene pool for Cd tolerance [29]. Here, 80 resequenced HMA3 homoeologs of Ae. tauschii were widely distributed and structured into six exons, and their sequences were highly homologous with one another (Fig 1). The sequence variation between them was concentrated more in the exonic than in intronic DNA (Fig 1). Ten haplotypes were revealed by these sequences and collapsed into ten distinct polypeptides (Fig 2). The structural organization of the HMA3 homoeologs shared conserved transmembrane domains (TM1 to 8), where 6 of the eight altered amino acids were involved in the interval between transmembrane domains, and the remaining 2 were prospectively involved in TM1 and TM4. Furthermore, variation in the BrHMA3 coding sequence is a key determinant of Cd translocation to and accumulation in the leaves of B. rapa [32]. Mutation at the 80th amino acid (R80H) in front of TM1 [5] and mutation in the 380th amino acid in TM6 from Ser to Arg [6] altered OsHMA3 function (S1 Fig). Moreover, the bread wheat cultivars JiMai22 and HeNong6425 and the US wheat cultivar Fielder showed 3-7 times higher Cd translocation than the rice cv. Nipponbare. Overexpression of rice OsHMA3 in wheat greatly decreases the Cd accumulation in grains [11]. Therefore, the altered peptide sequence revealed from Ae. tauschii has potential value for low/no Cd concentration wheat breeding.